This invention relates to novel hyaluronic acid associates (complexes) of 3d metal ions of the 4th period of the Periodic Table, with a stoichiometric composition, prepared through the interaction of equivalent amounts of an alkali metal hyaluronate, alkaline earth metal hyaluronate, silver hyaluronate or a quaternary ammonium hyaluronate and 3d metal ions in aqueous solution, and to pharmaceutical compositions containing these associates (complexes) as active ingredients.
The invention further relates to a process for the preparation of these novel associates (complexes) and compositions containing these associates (complexes) as active ingredients.
According to a particularly preferred embodiment of the process of the present invention, the aqueous solutions containing the novel hyaluronic acid associates (complexes) of 3d metal ions of the 4th period of the Periodic Table, with a stoichiometric composition, prepared through the interaction of equivalent amounts of sodium hyaluronate, and 3d metal ions in aqueous solution, are prepared directly from an aqueous solution of sodium hyaluronate.
The novel associates according to the present invention mainly involve zinc and cobalt hyaluronate. The compositions containing these latter associates may be pharmaceutical (therapeutical) or cosmetic and optionally other compositions. The compositions containing the novel associates according to the invention are therapeutically effective for e.g.: the acceleration of epithelization of epithelium-deficient body surfaces; healing of crural ulcers, decubitus (bed-ulcers), primarily not healing wounds, burns, radiation- or heat-induced wounds, vulgar acne and conglobated acnes, although they can be used in other areas, too.
The novel hyaluronate associates have been found to possess antibacterial activity. These hyaluronate associates have been found especially to possess activity against a number of bacteria that often infect crural or decubitus ulcers. For instance the new zinc hyaluronate complexes have been found to be highly effective against Staphylococcus aureus and Salmonella sp. The new cobalt hyaluronate complexes have been found to be highly effective against Staphylococcus aureus and E. coli. 
Furthermore the new hyaluronates have utility in ophthalmic preparations. The zinc hyaluronates have been found to be especially useful in this regard. The new zinc hyaluronates may be applied to the eye as topical antibacterial agents or they may be applied intraocularly as antibacterials. The zinc hyaluronates have been found to be especially effective against Staphylococcus aureus and against Pseudomonas aeruginosa, two types of bacteria that are implicated in eye infections. The most frequent eye contaminant is the Staphylococcus group, but the less frequent Pseudomonas aeruginosa group, is very dangerous and can cause complete blindness in 24 to 48 hours. The fact that the hyaluronate complexes are effective against both of these microorganisms is highly important.
Hyaluronic acid is a macromolecule known for more than 50 years and which has first been described by Meyer et al. [J. Biol. Chem. 107, 629 (1954); J. Biol. Chem. 114, 689 (1936)]. The structure determination was performed by Weissman et al. [J. Am. Chem. Soc. 76, 1753 (1954)]. Hyaluronic acid is a highly viscous native glucosaminoglycan containing alternating xcex21-3 glucouronic acid and xcex21-4 glucosamine moieties; its molecular weight is between 50000 and several (8 to 13) millions. The recovery of hyaluronic acid is an old task. The separation and use of an extra pure hyaluronic acid are described e.g. in the U.S. Pat. Nos. 4,141,973 and 4,303,676 and in the European Patent No. 0 144 019.
Until recently hyaluronic acid has been employed as the sodium salt e.g. in therapy, mainly in opthalmology, surgery and cosmetics. The salts of hyaluronic acid formed with alkali, alkaline earth, magnesium, aluminum, ammonium or substituted ammonium ions may serve as carriers for promoting the absorption of drugs (see the Belgian Patent Specification No. 904,547). Heavy metal salts of hyaluronic acid (wherein xe2x80x9cheavy metalsxe2x80x9d mean the elements of the 5th, 6th and 7th periods of the Periodic Table as well as the lanthanides and actinides) and within these the silver salt are utilized as fungicidal agents whereas the gold salt is employed for the treatment of arthritis (see the patent specification WO 87/05517).
It has been proven by various structure-elucidating methods that the secondary structure, i.e. the conformation of hyaluronic acid is changed by binding metal ions [W. T. Winter and A. Struther: J. Mol. Biol. 517, 761 (1977); J. K. Sheehan and E. D. T. Atkins: Int. J. Biol. Macromol. 5, 215 (183); and N. Figueroa and B. Chakrabarti: Biopolymers 17, 2415 (1978)]. Significantly varying effects on the molecular structure can be exerted even by metal ions of similar character as shown by comparative X-ray study of potassium and sodium hyaluronate [A. K. Mitra et al.: J. Macromol., Sci. Phys. 824, 1 and 21 (1985)]. This is all the more valid for compounds of hyaluronic acid formed with metal ions of various sorts bearing various charges.
No reference relating to hyaluronic acid associates (complexes) of 3d metal ions of the 4th period of the Periodic Table, with a stoichiometric composition, prepared through the interaction of equivalent amounts of an alkali metal hyaluronate, alkaline earth metal hyaluronate, silver hyaluronate or a quaternary ammonium hyaluronate and 3d metal ions in aqueous solution, can be found in the literature. Actually, according to gel filtration chromatography examinations, hyaluronic acid, in contrast with heparin, is unable to bind zinc ions [(R. F. Parish and W. R. Fair: Biochem. J. 193, 407 to 410 (1981)].
In spite of the fact that, according to the literature, hyaluronic acid (or its sodium salt) is unable to bind zinc ions, we undertook to investigate the coordination chemistry of the interaction between hyaluronic acid and 3d metal ions of the 4th period of the Periodic Table and among these, chiefly, zinc and cobalt ions. Since hyaluronic acid is nearly exclusively commercialized as its sodium salt thus being the basic substance of all systems containing hyaluronate, our investigations were begun on the interaction of sodium ions and hyaluronate. For this purpose the free sodium ion activity of aqueous sodium hyaluronate solutions was measured by using a sodium selective glass electrode. It was unambiguously found from these measurements that not more than 60% of sodium ions introduced as equivalent together with the carboxylate groups of hyaluronate are present as free ions in the aqueous solutions whereas the remainder of 40% is in a form bound to the hyaluronate.
According to our measurements, by increasing the sodium ion concentration the amount of the sodium ions bound can be raised to 50-55% calculated for all available carboxylate groups. Thus, it has been verified that, as contrasted with common properties of salts, sodium hyaluronate is not completely dissociated in aqueous solution.
There are several references which disclose the importance of Zn2+in the eye as well as in other parts of the human body. For instance see xe2x80x9cZinc in the Eyexe2x80x9d, Surv. Ophthalmol., September-October 1982 27(2), pp 114 to 122 and xe2x80x9cZinc Uptake in Vitro by Human Retinal Pigment Epitheliumxe2x80x9d, ophthalmology, June 1992, 99 (6), p. 841.
It is also known that bacterial contamination of the eye is a serious health problem. Reference is made to Remington""s Pharmaceutical Sciences, 18th Ed., p. 1588 (1980) which discusses the contamination of eye solutions and emphasizes that the most frequent contaminant is the Staphylococcus group. Less frequently the contaminant is Pseudomonas aeruginosa which is very dangerous to the eye. Infection of the eye with these bacteria can result in complete blindness over a 24 to 48 hour period.
Furthermore sodium hyaluronates have intraocular utility in the field of ophthalmology. Sodium hyaluronate can be injected into the eye during eye surgery and it is then washed out of the eye after the operation. See the Czech Inventor""s Certificate 264,719. There is no suggestion in any of the prior art references to prepare stoichiometric zinc hyaluronate nor to use zinc hyaluronate in the field of ophthalmology to treat bacterial infections.
U.S. Pat. No. 4,623,539 discloses hyaluronic acid compositions for coating the small intestines of mammals to limit absorption of food and thereby facilitate weight control. There is no mention of antibacterial activity for the compositions or of using the compositions for the treatment of crural or decubitus ulcers.
In the reference hyaluronic acid is mentioned among many cationic polymers. Hyaluronic acid is of course an amino polysaccharide. The cationic polymers specifically disclosed as same and exemplified in U.S. Pat. No. 4,632,539 that form complexes with zinc cations or alkaline earth metal cations all contain a sulfate group. It is emphasized that there is no disclosure of a single specific hyaluronic acid complex of any divalent metal cation in the reference.
It is clear from the three generic formulae at the top of col. 7 of the reference that zinc cations or alkaline earth metal cations are applied to amino polymers to ensure a cationic character only in the case where a sulfate group is present since the sulfated amino polymers are the only amino polymers disclosed therein that fall within a generic formula containing divalent cations. The two other generic formulae that encompass hyaluronic acid and the other amino polymers that do not contain sulfate groups include no divalent metal cations. The hyaluronic acid and the other amino polymers containing no divalent metal cation derive their cationic character when a proton is bonded to the amino nitrogen atom.
The presently disclosed zinc or cobalt hyaluronate complexes, unlike the cationic complexes of U.S. Pat. No. 4,623,539, have a neutral character, that is there is no extra positive charge in the present complexes, so they cannot be regarded as cationic.
In the next step of our investigations an aqueous solution of sodium hyaluronate was titrated with zinc chloride solution by using a sodium ion-selective electrode as mentioned above for following the change in the activity of free sodium ions in the system. A characteristic curve reflecting the process is shown in FIG. 1. It is perceivable that sodium ions originally bound to hyaluronate are liberated on the effect of zinc ions.
Based on the results of these measurements the total sodium ion concentration is liberated by an equivalent amount of zinc, a fact unequivocally proving that zinc ions are more strongly bound to hyaluronate than are sodium ions. Thus, the earlier statement that hyaluronic acid would be unable to bind zinc ions [R. F. Parrish and W. R. Fair: Biochem. J. 193, 407 (1981)] has experimentally been refuted.
Thereby, knowledge previously held by workers skilled in the art was disproved.
From our investigations discussed above it became clear that, through the interaction of equivalent amounts of sodium hyaluronate and zinc ions (zinc chloride) in aqueous solution a zinc hyaluronate associate with a stoichiometric composition is formed. After an appropriate isotonization the solution obtained can directly be used for therapeutical purposes and the zinc compound need not be prepared in solid state in a separate process. Preliminary examinations carried out by using cobalt ion and other 3d metal ions led to similar results.
Nevertheless, the complex was prepared in a solid state for characterization and the direct environment of the zinc ion was determined by using the xe2x80x9cExtended X-ray Absorption Fine Structurexe2x80x9d (EXAFS) method. It has been found that zinc is surrounded by four oxygen atoms in the first coordination sphere. The length of the Znxe2x80x94O bond distances is 199 pmxc2x11.5 pm whereas two carbon atoms are present in a longer distance of 241 pmxc2x115 pm from the zinc atom.
According to our examinations zinc hyaluronate significantly differs from the analogous copper complex which latter contains four equatorial and two axial Cuxe2x80x94O bonds with the values of 194 and 234 pm, respectively. The distance between the copper atom and the new two carbon atoms is 258 pm. The structure of the cobalt (II) complex is similar to the zinc complex but not to the copper complex; specifically the Coxe2x80x94O bond distance is 197 pmxc2x11.0 and the Coxe2x80x94C is 239 pmxc2x115 pm.
The present invention relates to hyaluronic acid associates (complexes) of 3d metal ions of the 4th period of the Periodic Table, with a stoichiometric composition, prepared through the interaction of equivalent amounts of alkali metal hyaluronate, alkaline earth metal hyaluronate, silver hyaluronate or a quaternary ammonium hyaluronate and 3d metal ions in aqueous solution.
The invention further relates to a pharmaceutical composition containing as active ingredient hyaluronic acid associates (complexes) of 3d metal ions of the 4th period of the Periodic Table, with a stoichiometric composition, prepared through the interaction of equivalent amounts of an alkali metal hyaluronate, alkaline earth metal hyaluronate, silver hyaluronate or a quaternary ammonium hyaluronate and 3d metal ions in aqueous solution, optionally in admixture with a carrier and/or other active ingredients and/or additives.
According to another aspect of the invention, there is provided a process for the preparation of the novel associates (complexes) of the invention, with a stoichiometric composition, which comprises:
a) adding an aqueous solution containing the equivalent amount of a salt, preferably the chloride of one of the 3d metal ions of the 4th period of the Periodic Table, to an equivalent amount of an aqueous solution of sodium hyaluronate or to an equivalent amount of another salt (alkali or alkaline earth metal salt, optionally silver salt) of hyaluronate; or
b) dissolving an associate formed from hyaluronic acid with a quaternary ammonium salt in an aqueous suspension in a solvent couple containing the aqueous solution of an equivalent amount of a 3d metal ion of the 4th period of the Periodic Table and a solvent which is partially miscible with water, preferably n-butanol; then precipitating the hyaluronic acid associates (complexes) of 3d metal ions of the 4th period of the Periodic Table, as stoichiometric compositions, by an alkanol or alkanone in a known manner, or
separating the precipitate from the solution and then, if desired
drying it under mild conditions.
This process serves for the preparation of aqueous solutions containing as active ingredient a stoichiometric composition which is a zinc or cobalt (II) hyaluronate associate (complex) or a similar associate of a 3d metal ion of the 4th period of the Periodic Table, respectively. These solutions were in each case prepared by the direct reaction of the metal ion with the hyaluronate component. This method of preparation made unnecessary to previously separate the active ingredients mentioned above in a solid state. In the solution prepared by using the process of the invention the amount of free (metal-unbound) hyaluronate is negligible even in the presence of an equivalent amount of zinc. In the presence of an excess of zinc ions the formation of the zinc hyaluronate associate (complex) becomes quantitative.
In the course of preparation of the metal associates as discussed above the pH remains at a value of about 4.5 to 6.5. In the case of a 0.2% by weight/volume (wt./vol.) hyaluronate solution the pH reaches a value of 5.4 whereas in the case of 0.5% by wt./vol. the pH value is 5. When necessary, the pH of the latter system can be adjusted to a value of 5.5 to 5.6 by adding a few drops of isotonic sodium acetate solution.
Solutions of two sorts containing zinc hyaluronate as active ingredient have been prepared by using the process discussed above.
1. Zinc Hyaluronate Solution Made Isotonic by an Excess of Zinc Chloride
Taking into consideration that free zinc chloride alone may also preferably be used in the dermatology, the osmotic pressure of the zinc hyaluronate solution was adjusted to the isotonic value by using an excess of zinc chloride. The solution thus obtained did not contain any free (zinc-unbound) hyaluronate at all but an excess of zinc chloride was present in the system together with zinc hyaluronate.
2. Zinc Hyaluronate Solution Made Isotonic by a Monosaccharide or a Sugar Alcohol
For a therapeutic use wherein the presence of hyaluronate-unbound zinc ions is not indicated, the stoichiometric solution containing zinc ions in an amount equivalent to the hyaluronate was made isotonic by using a polyalcohol (sugar alcohol, preferably sorbitol) or a mono- or disaccharide (preferably glucose). The free zinc ion and free hyaluronate content of these latter systems did not reach 5% of the total zinc or total hyaluronate content, respectively.
In the course of utilizing the associates according to the invention ion-free compositions may eventually be required. Namely, the associates prepared according to the above process of the invention usually contain sodium chloride or another salt formed from the starting hyaluronate cation and the anion of the 3d metal salt.
Two different process variants can be used for the preparation of a salt-free hyaluronic acid associate formed with a 3d metal ion. These are as follows.
a) A solution of a quaternary ammonium salt is portionwise added to the solution of a known hyaluronate, preferably sodium hyaluronate. After a satisfactory purification, the novel quaternary ammonium hyaluronate associate precipitated is dissolved under vigorous stirring in a solvent couple consisting of an aqueous solution of a 3d metal ion of the 4th period of the Periodic Table and a solvent which is partially miscible with water, preferably n-butanol. The two phases are allowed to separate, then the hyaluronate associate is precipitated by adding an alkanol or alkanone to the aqueous phase, the precipitate is separated and washed; or
b) after adding 2.0 to 3 volumes of a C1-3alkanol or C3-4alkanone under stirring to a zinc hyaluronate solution, suitably to a not isotonized solution containing zinc chloride in an amount equivalent to the hyaluronate, the zinc hyaluronate precipitated is filtered and washed with the alkanol or alkanone, respectively used for the precipitation. When necessary, the zinc hyaluronate is dissolved in ion-free water and the precipitation is repeated.
When a solid ion-free zinc hyaluronate is needed, the precipitate is dried under reduced pressure under mild conditions. In the case of a demand for an ion-free zinc hyaluronate solution it is preferable to dissolve the zinc hyaluronate made free from the solvent. According to both process variants an ion-free solid or dissolved product is obtained with an optional purity depending on the quality of the starting zinc hyaluronate.
The structural formula for the new hyaluronic acid associates of 3d metal ions of the fourth period of the Periodic Table has now been found to be as follows: 
wherein
M is a divalent cation of a 3d metal of the fourth period of the Periodic Table, especially zinc or cobalt; and
n is an integer between 62 and 10,000.
It is noted that two dimer units of hyaluronic acid within the brackets in the abovementioned Formula (I) have a molecular weight of about 800 Daltons, and the range of molecular weights of sodium hyaluronate used to prepare the new complexes has been given as 50,000 to 8 to 13 million Daltons. Where xe2x80x9cnxe2x80x9d is 62 to 10,000 this corresponds to a molecular weight of 50,000 to 8 million Daltons.